In vitro protein synthesis using natural messenger RNAs requires the presence of initiation factor 3 (IF3). These observations apply to in vitro systems from mammals, plants, invertebrates, and single-celled organisms. I propose to elucidate the physical chemistry of procaryotic IF3 protein interactions with a variety of ribonucleic acids. My colleagues and I have already observed that IF3 is a strongly binding RNA unwinding protein, with sequence specificity for the trinucleotide AUG. It is now appropriate to measure the thermodynamic parameters of binding equilibrium of IF3 with a variety of regular and fluorescently modified oligonucleotide single strands, hairpins and duplexes. Titrations of RNA circular dichroism and fluorescence detected circular dichroism, and of protein extrinsic fluorescence will be analyzed for intrinsic binding constants, site sizes and cooperativity constants. The thermodynamic parameters of binding kinetics will be measured by observing stopped-flow circular dichroism and stopped-flow fluorescence of RNAs, and stopped-flow fluorescence of protein extrinsic fluorophores. IF3 effects on the equilibrium binding of various fluorescent oligonucleotides to ribosomes will be measured by observing RNA fluorescence and fluorescence detected circular dichroism. IF3 effects on binding kinetics will be monitored by observing RNA stopped-flow fluorescence.